Soil & Water Res.

, 11, 2016 (1): 37–43 Original Paper

doi: 10.17221/21/2015-SWR

Moisture Effect on Soil Humus Characteristics

in a Laboratory Incubation Experiment

Cuilan LI 1,2, Shuqing GAO2, Jinjing ZHANG 2, Lanpo ZHAO 2 and Lichun WANG 3
1
National Engineering Laboratory for Improving Quality of Arable Land, Institute of Agricultural
Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing, P.R. China;
2
College of Resource and Environmental Science, Jilin Agricultural University, Changchun,
P.R. China; 2Institute of Agricultural Resources and Environments, Jilin Academy
of Agricultural Sciences, Changchun, P.R. China

Abstract
Li C., Gao S., Zhang J., Zhao L., Wang L. (2016): Moisture effect on soil humus characteristics in a laboratory incuba-
tion experiment. Soil & Water Res., 11: 37–43.

A 180-day laboratory incubation experiment (30°C) was conducted to investigate the quantitative and qualitative
characteristics of humic fractions in a Mollisol at different moisture conditions. The soil moisture contents were
30, 60, and 250% field water-holding capacity (WHC), which represented the low, middle, and high moisture levels,
respectively. The results showed that the carbon contents of the total soil and corresponding humic fractions gener-
ally decreased with increasing soil moisture. A significant difference was observed between the 250% WHC and the
two other moisture levels. By contrast, the carbon content of the water soluble fraction significantly increased with
increasing soil moisture levels. The solid-state 13C nuclear magnetic resonance (NMR) spectra showed that the alkyl
C/O-alkyl C, aliphatic C/aromatic C, and hydrophobic C/hydrophilic C ratios were in the order of 250% WHC > 30%
WHC ≈ 60% WHC, 30% WHC ≈ 60% WHC > 250% WHC and 250% WHC > 60% WHC ≈ 30% WHC for humic acid,
and 250% WHC > 30% WHC ≈ 60% WHC, 60% WHC ≈ 250 % WHC > 30% WHC and 30% WHC ≈ 250% WHC >
60% WHC for humin, respectively. These results indicated that a high moisture level was unfavourable for the carbon
accumulation of the total soil and humic fractions, whereas it was favourable for the accumulation of water soluble
carbon. Although soil moisture levels had a distinct effect on the chemical composition of humic acid and humin,
the decomposition degree of the two humic substances components, as indicated by the alkyl C/O-alkyl C ratio, were
both higher at a high moisture level than at a low moisture level. Therefore, the lower soil organic carbon content
at a high moisture level than at a low moisture level can be ascribed to the higher water soluble carbon content and
larger decomposition degree of humic acid and humin in the former. Our results are important for understanding
the behaviour and mechanisms of humic substances at specific soil moisture conditions.
Keywords: humic acid; humin; humus composition; soil moisture; solid-state 13C NMR

Soil organic matter is a major soil component. It has
an important function in both soil fertility and the
ecological environment (Paul 2014). Approximately
60–70% of organic matter in soil is composed of humic
substances (Loffredo & Senesi 2006). As the refrac-
tory organic carbon form of soil, humic substances
play a vital role in the atmospheric CO2 sequestration
(Spaccini et al. 2002). According to their solubility
in aqueous solution at different pH values, humic
substances can be divided into three main fractions,
namely humic acid, fulvic acid, and humin.
The formation of humic substances (i.e. humifica-
tion) is primarily a microbially mediated process (Zech
et al. 1997). The humification process is generally
accompanied by changes in the amount and chemi-
cal structure of humic substances (Ikeya et al. 2004).
Therefore, the factors that control microbial activity,
such as temperature, pH, and C/N ratio, determine

37
Original Paper Soil & Water Res., 11, 2016 (1): 37–43
the quantity and quality of humic substances formed
in soil. Among these factors, soil moisture is a major
factor affecting microbial activity (Brockett et al.
2012). The optimum soil moisture content for microbial
activity is found at intermediate levels (Iqbal et al.
2009), i.e. 50–70% of water-holding capacity (Stres
et al. 2008). Previous studies mainly focused on the
impact of soil moisture content on the mineralization
process of soil organic matter (Hossain & Puteh
2013; Chen et al. 2014; Guo et al. 2014; Lu & Xu
2014), however, its effects on the amount and chemi-
cal composition of humic substances remain unclear.
Mollisol, which accounts for about 7% of the world’s
ice-free land surface, is generally recognized as an
inherently fertile and productive soil (Liu et al. 2012).
Mollisol is characterized by high content of organic
matter and humic substances with a higher aromatic
structure compared with other soil types (Glaser &
Amelung 2003). Moreover, a high aliphaticity has
also been observed in both humic acid and humin in
Mollisol (Li et al. 2015). Using sequential extraction
with base + urea and DMSO + H2SO4 solvent systems,
Song et al. (2011) showed that humin in Mollisol is
largely made up of biological molecules from plants
and microorganisms. Mollisol is mainly distributed in
the northeast region of China. The region has been a
key base for commodity grain production. However,
long-term intensive cultivation practices have led
to a remarkable decline in soil organic carbon in
Mollisol over the last decades. Therefore, increasing
carbon sequestration to sustaining soil productivity
in the Mollisol region is important (Liu et al. 2010).
The present study attempts to provide a preliminary
examination of the effects of different soil moisture
contents on the quantitative and qualitative charac-
teristics of humic fractions in a Mollisol of northeast
China at controlled incubation conditions based on a
combination of classical humus composition analysis
and solid-state 13C NMR spectroscopy.
MATERIAL AND METHODS
Soil samples. Surface soil samples (0–20 cm) were
collected in May 2013 from a maize (Zea mays L.)
field located at the Lishu County (43°16'N, 124°26'E),
Jilin Province, northeast China. The soil was classi-
fied as Mollisol (USDA Soil Taxonomy), Phaeozem
(FAO Soil Taxonomy), or Isohumosol (Chinese Soil
Taxonomy). The soil samples were air-dried, crushed,
and passed through a 2 mm sieve after removing
the visible plant residues. The physical and chemi-
38
doi: 10.17221/21/2015-SWR
cal properties of the soil were as follows: pH (H 2O)
5.4, moisture content 52.1 g/kg, field water-holding
capacity (WHC) 345.5 g/kg, total organic C (TOC)
11.1 g/kg, total N 1.11 g/kg, and C/N ratio 10.0.
Incubation experiment. Sieved soil samples (200 g)
were weighed into 250 ml plastic beakers. Distilled
water was added to bring the soil moisture contents
to 30, 60, and 250% WHC, which represented the low,
middle, and high (i.e. flooding condition with a 1 cm-
deep water layer above the soil surface) moisture levels,
respectively. The beakers were covered with plastic
films with needle holes to maintain aerobic condition
and placed in an incubator in a completely randomized
design. The incubation experiment was conducted at
30°C for 180 days. During the incubation period, the soil
moistures were held constant by periodically weighing
and spraying distilled water as necessary. At the end of
the incubation, three replicate soil samples from each
treatment were removed, air-dried, and sieved through
a 2 mm mesh sieve for subsequent analysis.
Humus composition analysis. Humus composi-
tion was analyzed according to the method described
by Zhang et al. (2010). Briefly, soil sample was first
soaked in distilled water at 70°C for 60 min to isolate
the water soluble fraction. The soil residue was then ex-
tracted using a mixture of 0.1 mol/l NaOH and 0.1 mol/l
Na4P2O7 at 70°C for 60 min. The dark brown alkaline
supernatant solution, corresponding to the total humic
extract, was acidified with 0.5 mol/l H2SO4 solution to
coagulate the humic acid fraction, with the fulvic acid
fraction remaining in the supernatant solution. The
solid residue after alkaline extraction was referred to
as mineral-bound humin fraction. The carbon contents
of water soluble fraction (WSFC), total humic extract
(HEC), and humic acid fraction (HAC) were determined
by dichromate oxidation method. The carbon contents
of fulvic acid fraction (FAC) and mineral-bound humin
fraction (MHUC) was calculated by subtracting the
HAC from the HEC and subtracting the sum of WSFC
and HEC from the TOC, respectively.
Isolation of humic acid and humin. Humic acid
and humin were isolated at room temperature accord-
ing to the procedure described previously (Zhang
et al. 2013). Briefly, 20 g of soil sample from each of
the three replicates was first suspended in distilled
water and 0.05 mol/l HCl solution to remove the
poorly decomposed light fractions and carbonates,
respectively. The soil residue was then extracted with
200 ml of 0.1 mol/l NaOH and 0.1 mol/l Na 4P2O7 for
48 h. The extraction procedure was repeated eight
times until the supernatant was almost colourless.
Soil & Water Res., 11, 2016 (1): 37–43 Original Paper

doi: 10.17221/21/2015-SWR

The humic acid fraction was precipitated by acidifying
the alkaline supernatants to pH 1.0 using 6 mol/l HCl.
After three cycles of dissolution in 0.1 mol/l NaOH
and coagulation again in 6 mol/l HCl, the humic acid
precipitate was demineralized with 0.5% HCl-HF
solution. The alkaline-insoluble humin fraction was
treated ten times with 10% HF-HCl solution to re-
move mineral impurity. The resulting humic acid
and humin were dialyzed against distilled water, and
then freeze-dried.
Solid-state 13C NMR spectroscopic analysis of
humic acid and humin isolated. The solid-state 13C
cross-polarization magic-angle-spinning and total-
sideband-suppression (CPMAS TOSS) NMR spectra
were recorded with a Bruker AVANCE III 400 WB spec-
trometer (Bruker BioSpin AG, Fällanden, Switzerland)
at 100.6 MHz. From 4000 to 10 000 scans with 2048 data
points were collected over a spinning speed of 8 kHz, a
spectral width of 50 kHz, an acquisition time of 20 ms,
a recycle delay of 3 s, and a contact time of 2 ms. All the
free induction decays were transformed by applying a
zero filling of 16 384 and a line broadening of 80 Hz.
Chemical shifts were referenced externally to the meth-
ylene resonance of adamantane standard at 38.4 ppm.
The spectra were divided into four common chemical
shift regions representing alkyl C (0–50 ppm), O-alkyl C
(50–110 ppm), aromatic C (110–160 ppm), and carbonyl
C (160–210 ppm). Moreover, the O-alkyl C was divided
into regions of methoxyl C (50–60 ppm), carbohydrate
C (60–95 ppm), and di-O-alkyl C (95–110 ppm); and
aromatic C into regions of aryl C (110–145 ppm) and
phenolic C (145–160 ppm). The relative intensity of
each chemical shift region was obtained by integration
using the MestReNova software package (Version 5.3.1).
Statistical analysis. The soil humus compositions
among different moisture treatments were compared
using one-way ANOVA followed by LSD test at P < 0.05
(SPSS 16.0).
RESULTS AND DISCUSSION
Total organic carbon content in the soil. The TOC
content in the soil at different moisture treatments
is shown in Table 1. The TOC content gradually
decreased with the increase of soil moisture from
30 to 250% WHC. A significant difference was ob-
served between the 250% WHC and the two other
moisture treatments. The results suggested that a
high moisture level was unfavourable to the organic
carbon accumulation with respect to the low and
middle moisture levels. Previous studies have also
reported that organic carbon mineralization en-
hanced with increasing soil moisture from 20 up to
60–80% WHC (Iqbal et al. 2009; Chen et al. 2014;
Guo et al. 2014), which was consistent with our
results. However, there is currently no agreement
on the effects of flooding condition on soil organic
carbon mineralization. Some studies have shown that
flooding can promote organic carbon mineralization
(Smith 2005; Lu & Xu 2014), whereas other studies
have found that flooding inhibited SOC mineraliza-
tion (Iqbal et al. 2009; Hossain & Puteh 2013).
As indicated above, the present study showed that
flooding condition resulted in a higher SOC loss. The
reason for this is explained in the following sections.
Soil humus composition. The carbon contents of
humic fractions in soil at different moisture treat-
ments are also shown in Table 1. The WSFC, which
accounted for 0.92–1.80% of TOC, significantly in-
creased with increasing moisture levels, which sug-
gested that the accumulation of the water soluble
fraction was more favourable at a high moisture
level. Dissolved organic carbon is an easily avail-
able carbon source to soil microbial communities
(Straathof et al. 2014). Previous studies showed
that the influence of flooding on soil organic carbon
content depended on the equilibration between the

Table 1. Carbon contents of the total soil and corresponding humic fractions under different moisture treatments (in g/kg)

Treatment
TOC WSFC HEC HAC FAC MHUC HAC/FAC
(% WHC)
30 9.77 ± 0.26a 0.09 ± 0.01c 3.99 ± 0.07a 1.71 ± 0.06a 2.28 ± 0.11a 5.68 ± 0.29a 0.75 ± 0.06a
a b b a a a
60 9.56 ± 0.19 0.11 ± 0.00 3.76 ± 0.03 1.62 ± 0.05 2.14 ± 0.02 5.70 ± 0.19 0.76 ± 0.03a
250 8.61 ± 0.10b 0.16 ± 0.01a 3.37 ± 0.02c 1.40 ± 0.05b 1.97 ± 0.07b 5.08 ± 0.08b 0.71 ± 0.05a

WHC – field water-holding capacity; TOC – total organic carbon; WSFC – carbon content of water soluble fraction;
HEC – carbon content of total humic extract; HAC – carbon content of humic acid fraction; FAC – carbon content of fulvic
acid fraction; MHUC – carbon content of mineral-bound humin fraction; mean values ± standard deviation of three replicates
are presented; values within the same column not followed by the same letter differ significantly (P < 0.05)

39
Original Paper Soil & Water Res., 11, 2016 (1): 37–43
enhancement of dissolved organic carbon dissolu-
tion and the inhibition of microbial activity (Hao
et al. 2011; Lu & Xu 2014). When the enhancement
of dissolved organic carbon dissolution was larger
than the inhibition of microbial activity, soil organic
carbon content decreased. Although the microbial
activity has not been determined, the higher WSFC
content can reasonably explain the lower TOC con-
tent at flooding condition in the present study. By
contrast, the HEC, HAC, FAC and MHUC, which
accounted for 39.2–40.9%, 16.3–17.5%, 22.4–23.4%,
and 58.2–59.6% of TOC, respectively, followed a
pattern similar to TOC. The HAC, FAC, and MHUC
also exhibited insignificant differences between the
30% and 60% WHC treatments. The results implied
that a high moisture level was also unfavourable for
the accumulation of humic fractions in soil.
Although no significant differences were observed
among the three moisture treatments, the HAC/
FAC ratio was lower at 250% WHC than at the other
two moisture treatments. This implied that a high
moisture level was favourable for the accumulation
of fulvic acid fraction. Tardy et al. (1997) specu-
lated that fulvic acid dominated in wet or humid
climates and non-oxygenated condition based on
the thermodynamic stability calculation, which was
consistent with our results.
Solid-state 13C NMR spectra of humic acid and
humin. The extraction yields of humic acid were
3.42, 3.33, and 2.63 g/kg, and those of humin were
11.8, 11.6, and 11.3 g/kg at 30, 60, and 250% WHC,
respectively. The solid-state 13C CPMAS TOSS NMR
spectra of humic acid and humin at different moisture
treatments are presented in Figure 1, and the relative
(a)
250% WHC
60% WHC
30% WHC
200 180 160 140 120 100 80 60 40 20 0 200 180 160 140 120 100 80
Chemical shift (ppm)
Figure 1. Solid-state 13C cross-polarization magic-angle-spinning and total-sideband-suppression (CPMAS TOSS) NMR
spectra of soil humic acid (a) and humin (b) under different moisture treatments; WHC – field water-holding capacity
40
doi: 10.17221/21/2015-SWR
intensities of different carbon functional groups are
shown in Table 2. All spectra exhibited major signal
peaks at approximately 25, 31, 33, 56, 72, 106, 129, 152,
and 173 ppm. The peak assignments were reported in
previous studies (Zhang et al. 2009, 2011, 2013). The
relative intensities of the carbon functional groups
between humic acid and humin were different. Humic
acid was predominated by the aromatic group, whereas
humin was predominated by the O-alkyl group. How-
ever, in some soils, alkyl C dominated over O-alkyl C and
aromatic C in humic acid (Luo et al. 2008; Fernandes
et al. 2010) and humin (Dai et al. 2001; Chen et al.
2007). Moreover, Nierop et al. (1999) reported that
O-alkyl C dominated over alkyl C and aromatic C in
humic acid. Therefore, further studies are needed to
elucidate the possible reasons that lead to the different
results. On average, compared with humic acid, humin
had larger proportions of carbohydrate C and di-O-
alkyl C, and smaller proportions of alkyl C, methoxyl C,
aryl C, phenol C, and carbonyl C. The alkyl C/O-alkyl C
and hydrophobic C/hydrophilic C ratios of humic acid
were higher, whereas the aliphatic C/aromatic ratio was
lower than those of humin. The larger values of alkyl
C/O-alkyl C, aliphatic C/aromatic C, and hydrophobic
C/hydrophilic C ratios generally indicated the higher
degrees of alkylation, aliphaticity, and hydrophobicity of
humic substances (Zhang et al. 2013). Therefore, our
results suggested that humic acid was more alkylated,
more hydrophobic, and less aliphatic than humin. The
higher aliphaticity for humin than for humic acid has
also been observed in previous studies (Simpson et
al. 2011).
The alkyl C for humic acid was in the order of
250% WHC > 60% WHC ≈ 30% WHC. The meth-
(b)
250% WHC
60% WHC
30% WHC
60 40 20 0
Chemical shift (ppm)
Soil & Water Res., 11, 2016 (1): 37–43 Original Paper

doi: 10.17221/21/2015-SWR

Table 2. Relative carbon distribution (%) in different regions of chemical shift in 13C cross-polarization magic-angle-spinning and
total-sideband-suppression (CPMAS TOSS) NMR spectra of soil humic acid and humin under different moisture treatments

Treatment Chemical shift regions (ppm) Alip/

A/O-A HB/HI
(% WHC) 0–50 50–60 60–95 95–110 110–145 145–160 160–200 Arom
Humic acid
30 19.8 9.72 11.5 4.77 29.9 6.71 17.7 0.76 1.25 1.29
60 19.8 10.2 10.9 5.68 29.7 7.47 16.2 0.74 1.25 1.32
250 20.0 9.69 10.3 4.43 31.2 7.88 16.4 0.82 1.14 1.45
Average 19.9 9.88 10.9 4.96 30.3 7.35 16.8 0.77 1.21 1.35
Humin
30 17.0 6.13 24.3 8.60 26.0 7.20 10.8 0.44 1.69 1.01
60 18.2 5.92 25.6 9.04 22.9 7.53 10.8 0.45 1.93 0.95
250 20.4 6.51 22.3 8.01 24.2 6.13 12.5 0.55 1.89 1.03
Average 18.5 6.19 24.1 8.55 24.4 6.96 11.3 0.48 1.83 0.99

WHC – field water-holding capacity; alkyl C/O-alkyl C = (0–50)/(50–110); aliphatic C/aromatic C = [(0–50) + (50–110)]/
(110–160); hydrophobic C/hydrophilic C = [(0–50) + (110–160)]/[(50–110) + (160–200)]

oxyl C, carbonhydrate C, and di-O-alkyl C in the
O-alkyl C region were in the order of 60% WHC >
30% WHC ≈ 250% WHC, 30% WHC > 60% WHC
> 250% WHC, and 60% WHC > 30% WHC > 250%
WHC, respectively. The aryl C and phenol C in the
aromatic C region were in the order of 250% WHC
> 30% WHC ≈ 60% WHC and 250% WHC > 60%
WHC > 30% WHC, respectively. The carbonyl C
was in the order of 30% WHC > 250% WHC ≈ 60%
WHC. The above changes in relative intensity of
the carbon functional groups resulted in the alkyl
C/O-alkyl C ratio in the order of 250% WHC > 30%
WHC ≈ 60% WHC, aliphatic C/aromatic C ratio in
the order of 30% WHC ≈ 60% WHC > 250% WHC,
and hydrophobic C/hydrophilic C ratio in the order
of 250% WHC > 60% WHC ≈ 30% WHC. The results
indicated that humic acid was more alkylated, more
hydrophobic, and less aliphatic at a high moisture
level than at the two other moisture levels.
The alkyl C for humin was in the order of 250%
WHC > 60% WHC > 30% WHC. The methoxyl C in
the O-alkyl C region was in the order of 250% WHC
> 30% WHC > 60% WHC, and the carbonhydrate C
and di-O-alkyl C were in the order of 60% WHC >
30% WHC > 250% WHC. The aryl C and phenol C
in the aromatic C region were in the order of 30%
WHC > 250 % WHC > 60% WHC and 60% WHC >
30% WHC > 250% WHC, respectively. The carbonyl
C was in the order of 250% WHC > 30% WHC >
60% WHC. The above changes resulted in the alkyl
C/O-alkyl C ratio in the order of 250% WHC >30%
WHC ≈ 60% WHC, aliphatic C/aromatic ratio in
the order of 60% WHC ≈ 250% WHC > 30% WHC,
and hydrophobic C/hydrophilic C ratio in the order
of 30% WHC ≈ 250% WHC > 60% WHC. These re-
sults indicated that humin was more alkylated and
more aliphatic at a high moisture level than at a low
moisture level, whereas the hydrophobicity degree
was similar at the two moisture levels.
The above results showed that although soil mois-
ture levels had a distinct effect on the chemical com-
position of humic acid and humin, the alkyl C/O-alkyl
C ratios of the two humic substance fractions were
all higher at a high moisture level than at a low soil
moisture level. The O-alkyl C was generally consid-
ered as an easily bio-decomposable organic com-
ponent. The greater value of the alkyl C/O-alkyl C
ratio thereby indicates that soil organic matter was
more decomposed (Chavez-Vergara et al. 2014;
Panettieri et al. 2014). In our study, the larger
alkyl C/O-alkyl C ratio of humic acid and humin at a
high moisture level than at a low moisture level can
explain their lower carbon contents in the former.
CONCLUSION
This study has shown that the difference in soil
moisture contents influenced the amounts and chemi-
cal composition of soil humic fractions. The increase
in soil moisture was favourable for the accumula-
tion of water soluble carbon within the range of the
experimental moisture levels, whereas it resulted

41
Original Paper Soil & Water Res., 11, 2016 (1): 37–43
in losses of humic fractions. Humic acid was more
alkylated, less aliphatic, and more hydrophobic,
whereas humin was more alkylated and more aliphatic
at a high moisture level than at a low moisture level.
The high water soluble carbon content and large
decomposition degree of humic acid and humin can
explain the low soil organic carbon content at a high
moisture level against a low moisture level.
Acknowledgements. This work was supported by the Na-
tional Natural Science Foundation of China (No. 41471196,
31470506), the National Key Technologies R&D Program of
China (2013BAC09B01, 2013BAD07B02), and the Fund of
National Engineering Laboratory for Improving Quality of
Arable Land (No. 201501).
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Received for publication January 30, 2015
Accepted after corrections May 19, 2015
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